The detection of target nucleic acid in test samples is important in various fields, including medicine and biology. Many compositions, assay platforms, and procedures are available for the detection of specific nucleic acid molecules. In order for detection to be reproducible and accurate, these procedures require no or low levels of non-specific background amplification. However, amplification methods may give rise to false positive signals that affect the quality, accuracy, reproducibility, and overall reliability of the results. In some assays these “false” positive signals can be detected in samples, including control samples that contain non-template DNA (non-target DNA) or even samples that lack any DNA template.
One common method used for amplification of specific sequences from a population of mixed nucleic acid sequences is the polymerase chain reaction (PCR). Since a typical PCR is carried out at three different temperatures, the reaction can be associated with challenges such as difficulty in maintaining accurate temperatures and that the time loss increases in proportion to the number of amplification cycles. The denaturation of a double-stranded template DNA into single strands (while dependent to some extent on the particular sequence) often requires the use of high “melting” temperatures, which limits the class of DNA polymerases that can be used to those that are highly thermostable. Consequently, isothermal amplification platform technologies have been developed to detect nucleic acids under reaction conditions that are milder than those used in PCR. Nevertheless, these isothermal amplification technologies have not addressed the challenges that are presented by non-specific amplification events and high background signals that can interfere with target sequence detection.
The following disclosure provides alternative methods and compositions for detecting a nucleic acid sequence (such as DNA or RNA) under reaction conditions that are less rigorous than those used in PCR. The methods and compositions maintain sequence selectivity and sensitivity that allow for the detection of nucleic acid molecules that may be in a sample at low concentrations and/or nucleic acid molecules of a short length. The methods and compositions also reduce any background signal that may result from non-specific and/or target-independent amplification events. Among other aspects, the disclosure provides novel methods and nucleic acid molecules that can improve the detection limit of target nucleic acids in a sample under low temperature, isothermal conditions, and can simplify or improve sample preparation and automated methods of detection.